The objective of the proposed research is to develop a living replacement of skin that can be used in the treatment of major burns and skin losses. New methods have recently been developed in the laboratory of one of the co-investigators for convenient and rapid growth in defined medium of human epidermal keratinocytes, and for control of keratinocyte differentiation in culture. In addition, a slowly biodegradable synthetic dermal membrane composed of crosslinked collagen and glycosaminoglycans has recently been described in the literature for use in treating major burns. The overall research plan is to establish cultures of epidermal keratinocytes from a small skin biopsy, expand the cells in culture, coat the membrane with cultured keratinocytes, induce stratification of the keratinocytes and terminal differentiation of the outer layer, and then use the living artificial skin that has been generated for grafting. The cultures will be carefully monitored at all stages to verify absence of malignant transformation and undesirable antigenic changes. An interdisciplinary research team of basic scientists and physicians has been assembled with all of the needed expertise in cell culture, dermatology, biochemistry, oncology and clinical management of burns needed for this project. We anticipate that this procedure will allow cells from a small amount of a burn patient's unburned skin to be expanded rapidly in number and used to prepare a graft that can then be placed permanently over a large burned area with minimum risk of rejection. Such a procedure should greatly reduce total treatment time for patients with burns over a large area of the body, reduce the amount of donor tissue required for grafting, and increase the chances of saving very severely burned patients.